The present invention relates to a method for performing rapid and high accuracy determination of a substrate in a sample in a simplified manner.
As the method for quantitative analysis of sugars such as sucrose and glucose, polarimetry, colorimetry, reductiometry, and methods using a variety of chromatography have been developed. However, all of those methods have poor accuracy because of poor specificity to sugars. Of those methods, polarimetry is simple in manipulation but is largely affected by the temperature during operation. Therefore, polarimetry is not a suitable method for ordinary people to carry out determination of sugars at home or elsewhere in a simplified manner.
Apropos, various types of biosensor have been developed recently which use specific catalytic actions of enzymes.
In the following, a method of glucose determination will be described as one example of the method of substrate determination in a sample solution. A generally known electrochemical method of glucose determination is a method which uses glucose oxidase (EC1.1.3.4; hereinafter abbreviated to GOD) and an oxygen electrode or a hydrogen peroxide electrode (for instance, xe2x80x9cBiosensorxe2x80x9d ed. by Shuichi Suzuki, Kodansha).
GOD selectively oxidizes a substrate xcex2-D-glucose to D-glucono-xcex4-lactone using oxygen as an electron mediator. Oxygen is reduced to hydrogen peroxide in the presence of oxygen in the course of oxidation reaction by GOD. A decreased amount of oxygen is measured by the oxygen electrode or, otherwise, an increased amount of hydrogen peroxide is measured by the hydrogen peroxide electrode. The decreased amount of oxygen or increased amount of hydrogen peroxide is proportional to the glucose content in the sample solution, so that glucose can be determined based on the decreased amount of oxygen or increased amount of hydrogen peroxide.
As can be speculated from the reaction process, this method has a drawback that the measurement result is largely affected by the oxygen concentration in the sample solution. Furthermore, measurement is impossible in the absence of oxygen in the sample solution.
Therefore, a novel type glucose sensor has been developed which does not use oxygen as the electron mediator but uses an organic compound or a metal complex including potassium ferricyanide, ferrocene derivatives, quinone derivatives, etc. as the electron mediator. This type of sensor oxidizes a reduced form electron mediator resulting from enzyme reaction on the electrode and determines glucose concentration contained in the sample solution based on the quantity of oxidation current. The use of such organic compound or metal complex as the electron mediator in place of oxygen enables formation of a reaction layer while exactly carrying a known amount of GOD and either of such electron mediators in a stabilized state. In this case, since the reaction layer can be integrated in an almost dry state with the electrode system, a disposable type glucose sensor based on this technology has been drawing much attention currently.
The disposable type glucose sensor facilitates measurement of glucose concentrations with a measurement device by simple introduction of a sample solution into the sensor detachably connected to the measurement device. Application of such technic is not limited only to glucose determination and can be extended to determination of other substrate contained in the sample solution.
Measurement using the sensor as described before can determine the substrate concentration based on a flowing oxidation current value resulting from oxidation of a reduced form electron mediator on a working electrode. However, when blood, a fruit juice or something like that is used as a sample, any easy-to-oxidize substance contained in the sample solution, such as ascorbic acid, uric acid, etc. is concurrently oxidized on the working electrode together with the reduced form electron mediator. Oxidation reaction of such easy-to-oxidize substance may sometimes affect the measurement result.
In addition, in the measurement using the sensor as mentioned above, a reaction producing hydrogen peroxide using dissolved oxygen as an electron mediator may proceed concurrently with the reduction of the carried electron mediator on the reaction layer. Furthermore, the hydrogen peroxide produced by the reaction reoxidizes the reduced form electron mediator. This may eventually produce a negative error in the measurement result due to the dissolved oxygen when the substrate concentration is to be measured based on the oxidation current of the reduced form electron mediator.
The above-mentioned method often applies a voltage between the working electrode and a counter electrode to detect liquid junction, namely, to detect supply of sample solution on the basis of an electrical change between the two electrodes prior to application of a voltage between the working electrode and the counter electrode in order to obtain a current response. At that time, it sometimes occurs that measurement starts before supply of sufficient amounts of sample solution to the electrode system due to a change in resistance value between the above-mentioned working electrode and the counter electrode, which may sometimes affect the measurement result. Induction of a change in the condition of an interface of the working electrode may also affect the measurement result.
Furthermore, a measurement method with a two-electrode system uses a counter electrode as a reference electrode. This causes a change in potential of the counter electrode as the standard in association with the oxidation-reduction reaction at the working electrode, which also affects the measurement result.
The object of the present invention is to eliminate inconveniences as described above and provide a method of determination facilitating accurate measurement of substrate concentration by removing influences of easy-to-oxidize substances.
Another object of the present invention is to provide a method of substrate determination with lesser variations in sensor response.
The present invention is a method for determining the concentration of a substrate in a sample solution using a biosensor comprising an electrically insulating base plate, an electrode system having a working electrode, a counter electrode and a third electrode to be used as an interfering substance detecting electrode, each being formed on the above-mentioned base plate, and a reaction layer which contains at least an oxidoreductase and an electron mediator and is formed on the electrode system omitting the third electrode, wherein the electron mediator is reduced by the generating electrons upon reaction between the substrate contained in the sample solution and the oxidoreductase to measure a reduced amount of the electron mediator electrochemically,
the method being characterized by comprising the following steps:
(a) a step of applying a voltage between the counter electrode and the third electrode;
(b) a step of supplying the sample solution to the reaction layer;
(c) a step of detecting an electrical change between the counter electrode and the third electrode due to supply of the sample solution to the reaction layer;
(d) a step of measuring a current flowing between the counter electrode and the third electrode after the above-mentioned detecting step (c);
(e) a step of releasing voltage application between the counter electrode and the third electrode after the above-mentioned measuring step (d);
(f) a step of applying a voltage between the working electrode and the counter electrode; and
(g) a step of subsequently measuring a current flowing between the counter electrode and the working electrode.
The present invention also provides a method for determining the concentration of a substrate in a sample solution using a biosensor comprising an electrically insulating base plate, an electrode system having a working electrode, a counter electrode and a third electrode to be used as an interfering substance detecting electrode, each being formed on the above-mentioned base plate, a reaction layer which contains at least an oxidoreductase and an electron mediator and is formed on the electrode system omitting the third electrode, and a cover member forming a sample solution supply pathway to introduce a sample solution from a sample solution supply port into the above-mentioned reaction layer on the above-mentioned base plate, the third electrode being located upstream of the sample solution supply pathway from the reaction layer, wherein the electron mediator is reduced by the produced electrons upon reaction between the substrate contained in the sample solution and the oxidoreductase to measure a reduced amount of the electron mediator electrochemically,
the method comprising the following steps:
(a) a step of applying a voltage between the counter electrode and the third electrode;
(b) a step of supplying the sample solution to the reaction layer;
(c) a step of detecting an electrical change between the counter electrode and the third electrode due to supply of the sample solution to the reaction layer;
(d) a step of measuring a current flowing between the counter electrode and the third electrode after the above-mentioned detecting step (c);
(e) a step of releasing voltage application between the counter electrode and the third electrode after the above-mentioned measuring step (d);
(f) a step of applying a voltage between the working electrode and the counter electrode; and
(g) a step of subsequently measuring a current flowing between the counter electrode and the working electrode.
For the method of determination in accordance with the present invention, the use of the third electrode as reference electrode is preferred. Namely, a voltage is also applied between the working electrode and the third electrode during the above-mentioned step (f).
When a biosensor with the cover member being integrally combined with the above-mentioned base plate is used, it is also preferable to provide a lecithin carrying layer on an exposed wall surface of the cover member to the sample solution supply pathway.
It is preferred that the above-mentioned reaction layer further contains a hydrophilic polymer.